1. Field of the Invention
The present invention relates to a method of detecting and enumerating sulphate-reducing bacteria.
2. Description of Related Art
Sulphate-reducing bacteria (SRB) use sulphate as electron acceptor under anaerobic conditions, via the anaerobic respiration of sulphates (energy reduction), to produce sulphides while recovering, during this reduction, the energy necessary for their growth. This metabolic characteristic constitutes a common characteristic of these organisms, regardless of their phylogenetic position (Legall and Fauques, 1988).
These bacteria are recognized to be the principal microorganisms responsible for the biological formation of hydrogen sulphide (H2S). This H2S of biological origin in particular, and the metabolism of sulphate-reducing bacteria in general, cause many problems for industrialists such as the biological corrosion of steel, on the one hand, and the potential risk for staff, on the other (Postgate, 1979). In the petroleum industry, in addition to the abovementioned pernicious effects, the sulphate-reducing bacteria are also involved in impairing the quality of crude oil (Cord-Ruwich et al., 1987). The detection of these sulphurogenic bacteria therefore constitutes a major challenge for combating the production of H2S in a large number of industrial activities (Tatnall et al., 1988).
Microbial culture techniques applied to the detection and to the enumeration of these microorganisms have been developed (API, 1982; Magot et al., 1988; Scott and Davies, 1992). These methods require an incubation time of 10 to 21 days and are therefore poorly suited to the monitoring of contaminations of fluids in real time. Alternative methods allowing rapid measurement of the level of contamination have also been developed, such as for example the xe2x80x9cRapid Check(trademark)xe2x80x9d from Conocco, based on the immunodetection of APS reductase (Horacek and Gawell, 1988, EP 272,916), or the xe2x80x9cHydrogenase test(trademark)xe2x80x9d (Caproco), which detects the activity of hydrogenases, enzymes which are present in the SRBs but are not specific to these organisms (Scott and Davies, 1992). However, none of these methods is sufficiently sensitive or specific.
The authors of the present invention have developed a method of detecting and enumerating sulphate-reducing bacteria which combines these two advantages: sensitivity and specificity and which combines, in addition, speed with reliability. They therefore directed their attention to the sulphate energy reduction pathway and more specifically to that of APS reductase.
APS reductase (or Adenylylsulphate reductase) which allows the reduction of adenosine phosphosulphate (APS) (product of the activation of sulphate by ATP sulphurylase), is a cytoplasmic enzyme containing two subunits (xcex1 and xcex2) known to be involved only in the anaerobic respiration of sulphate (Legall and Fauques, 1988). This enzyme is not therefore present in non-sulphate-reducing organisms since it is not involved in the assimilatory reduction which allows the incorporation of sulphur into various molecules necessary for life, such as amino acids and vitamins.
On the basis of two sequences of the gene encoding this enzyme deposited in data banks, one derived from an organism in the domain of Bacteria (Desulfovibrio vulgaris, em_ba: 269372) and the other from the sector of Archaea (Archaeoglobus fulgidus: em_ba: X63434), the authors of the present invention were able to amplify and sequence various genes encoding APS reductase. Surprisingly, they observed that this gene is remarkably well conserved whereas the phylogenetic diversity of the organisms studied could not a priori suggest it. This result opened the perspective for using this gene as a target for the specific detection of sulphate-reducing bacteria, especially in the domain of Bacteria.
The subject of the present invention is therefore the use of at least one nucleotide sequence which hybridizes specifically with a fragment of the APS reductase gene or a fragment of the mRNA transcribed from the APS reductase gene to detect the presence of sulphate-reducing bacteria in a sample.
The subject of the present invention is more particularly a method for the specific, qualitative or quantitative detection of sulphate-reducing bacteria in a sample which is likely to contain them, the said method comprising the extraction of the DNA or of the RNA from the said sample and the detection of at least one fragment of the APS reductase gene or one fragment of the mRNA transcribed from the APS reductase gene, an indicator of the presence of sulphate-reducing bacteria in the said sample.
The extraction of the DNA or of the RNA from the said sample may be carried out by standard techniques which are well known to persons skilled in the art.
More particularly, the detection of at least one fragment of the APS reductase gene comprises the specific gene amplification of at least one fragment of the gene for the xcex1 subunit of APS reductase. Advantageously, the gene amplification products may, in addition, be subjected to hybridization with a probe which is specific for the said fragment of the gene for the xcex1 subunit of APS reductase, the said probe being labelled in a detectable manner.
According to another embodiment, the detection of at least one fragment of the APS reductase gene comprises the hybridization of the extracted DNA with a probe which is specific for the said fragment of the gene for the xcex1 subunit of APS reductase, the said probe being labelled in a detectable manner.
According to another embodiment, the method of the invention comprises the extraction of the RNA from a sample which is likely to contain sulphate-reducing bacteria and the detection of at least one fragment of the mRNA which is transcribed from the APS reductase gene.
In this case, the detection may be carried out by direct hybridization of a specific nucleotide probe labelled in a detectable manner with the extracted mRNA, and/or by specific amplification of the mRNA encoding APS reductase, in particular by RT-PCR (reverse transcription followed by a polymerase chain reaction).
The subject of the present invention is also an oligonucleotide having a nucleotide sequence which is essentially identical to a sequence chosen from the sequences SEQ ID No. 1 to 25. Such an oligonucleotide is in particular useful as a primer for amplifying a fragment of the gene for the xcex1 subunit of APS reductase, or as a probe which hybridizes with a fragment of the gene for the xcex1 subunit of APS reductase or the product of amplification thereof.
Preferably, it is possible to use as a primer an oligonucleotide having a sequence which is essentially identical to one of the sequences SEQ ID No. 11 to 18, and as a probe an oligonucleotide having a sequence which is essentially identical to one of the sequences SEQ ID No. 19 to 25.
xe2x80x9cEssentially identicalxe2x80x9d is understood to mean that the sequence of the oligonucleotide is identical to one of the sequences SEQ ID No. 1 to 25 or that it differs from one of these sequences without affecting the capacity of these sequences to hybridize with the gene for the xcex1 subunit of APS reductase. A sequence which is xe2x80x9cessentially identicalxe2x80x9d to one of the sequences SEQ ID No. 1 to 25 may in particular differ therefrom by a substitution of one or more bases or by deletion of one or more bases located at the ends of the oligonucleotide, or alternatively by addition of one or more bases at the ends of the oligonucleotide. Preferably, such an oligonucleotide has a minimum size of 10 nucleotides, preferably of at least 14 nucleotides.
According to a preferred embodiment of the invention, the method of detecting sulphate-reducing bacteria in a sample which is likely to contain them according to the invention advantageously comprises the steps consisting in:
extracting the DNA from the said sample;
bringing the DNA extracted in step i) into contact with at least one primer consisting of an oligonucleotide having a nucleotide sequence which is essentially identical to a sequence chosen from the sequences SEQ ID No. 1 to 25, preferably No. 1 to 18, under conditions allowing the specific amplification of a fragment of the gene for the xcex1 subunit of APS reductase which may be present in the DNA extract;
bringing the product of amplification into contact with a probe consisting of an oligonucleotide having a nucleotide sequence which is essentially identical to a sequence chosen from the sequences SEQ ID No. 1 to 25, preferably No. 19 to 25, the said probe being labelled in a detectable manner, under conditions allowing the specific hybridization of the said product of amplification and the said probe;
detecting the hybridization complex formed between the product of amplification and the said probe, an indicator of the presence of sulphate-reducing bacteria in the sample.
xe2x80x9cConditions allowing the specific amplificationxe2x80x9d is understood to mean conditions of temperature, of reaction time and optionally the presence of additional agents which are necessary for the fragment of the gene for the xcex1 subunit of APS reductase, to which the primers as defined above have hybridized, to be copied identically.
Preferably, the amplification method used is a polymerase chain reaction (PCR) which is well known to persons skilled in the art (Sambrook et al., 1989), which uses a pair of primers as defined above.
xe2x80x9cConditions allowing the specific hybridizationxe2x80x9d is understood to mean high stringency conditions which prevent the hybridization of the oligonucleotide with sequences other than the gene for the xcex1 subunit of APS reductase.
The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
For the sequences comprising more than 30 bases, Tm is defined by the relationship: Tm=81.5+41(%G+C)+16.6 Log(concentration of cations)xe2x88x920.63(%formamide)xe2x80x94(600/number of bases) (Sambrook et al., Molecular Cloning, A laboratory manual, Cold Spring Harbor laboratory Press, 1989, pages 9.54-9.62).
For the sequences of less than 30 bases in length, Tm is defined by the relationship:
Tm=4(G+C)+2(A+T). 
Under appropriate stringency conditions, at which the aspecific sequences do not hybridize, the hybridization temperature is approximately 5 to 30xc2x0 C., preferably 5 to 10xc2x0 C. below Tm, and the hybridization buffers used are preferably solutions with a high ionic strength such as a 6xc3x97SSC solution, for example.
The oligonucleotide probes used in the method of the invention are labelled in a detectable manner. For that, several techniques are accessible to persons skilled in the art, such as for example fluorescent, radioactive, chemiluminescent or enzymatic labelling.
An internal amplification control may be advantageously used in order to avoid an ambiguous interpretation of negative results of the amplification method. Indeed, for example, an absence of amplification by PCR may be due to problems of inhibition of the reaction or to the absence of a target.
The authors of the present invention propose using, as an internal control, a plasmid including oligonucleotide sequences which allow the amplification of a fragment of the APS reductase gene, the said oligonucleotide sequences flanking a sequence differing from the said fragment of the APS reductase gene by its size and/or its sequence. The said oligonucleotide sequences which are specific for a fragment of the APS reductase gene may be chosen in particular from the sequences SEQ ID No. 1 to 25, preferably the sequences No. 11 to 18. An example of such a plasmid is represented in FIG. 4.
Added in a limiting concentration to the PCR reaction mixture, this plasmid allows the amplification of a DNA fragment of 289 bp (base pairs) when no specific target is present in the sample. Thus, in the example selected, the presence of a fragment of 289 bp, without a fragment of 205 bp, indicates the functioning of the reaction and the absence of a specific target, that is to say of sulphate-reducing bacteria from the sample studied.